The present invention relates to a promoter which is active in the vascular endothelium and to its use for expressing genes of interest in this tissue.
The vascular endothelium is a cell monolayer which is formed from approximately a million endothelial cells which are distributed throughout the organism.
The endothelium controls the permeability of the blood vessels to fluids and to blood cells and regulates haemostasis and thrombosis. Change in vascular homeostasis can lead to serious pathologies such as arteriosclerosis, which is a major factor in cardiovascular diseases which constitute the prime cause of death in the Western countries, or the uncontrolled vascular proliferation which is observed, for example, in inflammatory diseases (rheumatoid arthritis), diabetic retinopathy, neoplastic angio-genesis or vascular tumours (angiosarcomas, Kaposi""s sarcoma).
There is a large number of possible applications for modulating the responses of the endothelial cells by directing expression of a gene of interest in these cells, with examples of these applications being as follows:
in the case of cardiovascular diseases, direct synthesis by the blood vessel wall of fibrinolytic agents such as the plasminogen activators: uPA and tPA;
controlling vascular proliferation:
either in order to stimulate it, for example in order to recreate an antithrombotic surface after denudation of the blood vessel (arteriosclerosis), and to avoid a restenosis following angioplasty, or in order to promote revascularization of the ischaematized tissues;
or in order to slow it down, for example in order to inhibit neoplastic progression by destroying the vascularization of the cancerous tissues. This can be achieved, for example, by constructing dominant-negative mutants for growth factor receptors (flk-l, flt-1, etc.), or by using antisense RNA.
Furthermore, since the vascular endothelium is in direct contact with the blood, it is readily accessible to gene vectors which are introduced by the venous route, and the products of the genes which it expresses can be secreted directly into the circulating blood. These properties can be exploited in order to achieve the secretion of proteins (coagulation factors, hormones, etc.) into the blood circulation.
Furthermore, on account of their presence in the capillaries, in the whole of the body, the vascular endothelial cells represent a particularly advantageous host for expressing, close to their site of action, cell effectors such as, for example, cytokine receptors or competitors of these receptors.
Finally, the endothelial cells have a lifetime which, in man, can amount to as much as 5 to 20 years [FAN et al.; TIPS, 16, p. 57, (1995)]. Transduced endothelial cells can therefore survive and express a transgene for a relatively long period of time.
Studying the in vivo functions of the vascular endothelium, as well as modifying these functions for a therapeutic purpose, requires the use of animal models which enable the role of each of the proteins expressed by the endothelial cells to be assessed individually, whether it is a matter of overexpressing or under-expressing a protein which is produced naturally by these cells, of assessing the activity of novel, potentially therapeutic molecules, or of studying the functional consequences of expressing a heterologous protein.
In order to be in a position to use effectively the potential advantages offered by transferring genes into the vascular endothelium, it is necessary to have available vectors which make it possible to obtain a stable expression of a gene of interest in the endothelial cells, both in vitro and in vivo; furthermore, this expression should, in certain cases, be specific for this tissue in order to avoid the problems which could result from ubiquitous expression.
A certain number of proteins are currently known which are expressed more or less specifically in the endothelium and whose promoters represent potential candidates for expressing a heterologous gene in a tissue-specific manner: the von Willebrand factor [FERREIRA et al., Biochem. J. 293, p. 641-648, (1993)], PECAM-1 (CD31) [De LISSER et al., Immunol. Today, 15, p. 490, (1994)], preproendothelin 1 [HARATS et al., J. Clin. Invest. 95, p. 1335, (1995)], integrin xcex1vxcex23 [BROOKS et al., Science, 264, p. 569, (1994)], P selectin [PAN and McEVER, J. Biol. Chem. 268, p. 22600, (1993)], VE cadherin [LAMPUGNANI et al., J. Cell. Biol. 118, p. 1511-1522, (1992); BREIER et al., Blood, 87, p. 630-641, (1996)], E selectin [WHITLEY et al., Mol. Cell. Biol., 14, p. 6464, (1994)], vascular endothelial growth factor receptor (Flk-1 or KDR) [MILLAUER et al., Cell. 72, p. 835-846, (1992)] and a tyrosine kinase receptor termed tie-2 [SCHLAEGER et al., Development, 121, p. 1089-1098, (1995)].
Some of the promoters which regulate the expression of the above genes have been cloned and studied in vitro in cell cultures, in particular those for E selectin, the von Willebrand factor and preproendothelin 1 [INOUE et al., J. of Biol. Chem. 264, p. 14954-14959, (1989)]. Even if consensus sequences for binding known transcription factors have been identified in some of these promoters, no sequence responsible for the endothelial specificity of the expression has yet been characterized.
For example, in the case of the preproendothelin promoter, an in vivo expression is observed which is not limited to the endothelium but is found in other cell types such as respiratory and intestinal epithelium and the cells of the vascular tunica media [HARATS et al., J. Clin. Invest., 95, p. 1335, (1995)].
Moreover, in a large number of cases, the observations made in vitro do not reflect the true activity and specificity of the promoter sequence in vivo, especially since it has been reported that, in the case of a heterologous gene, this activity and specificity can differ from those observed with the endogenous gene.
For example, the influence of the tie-2, von Willebrand factor and preproendothelin promoter regions on expression of a reporter gene has been studied in vivo in transgenic mice.
Thus, SCHLAEGER et al., [Development 121, p. 1089-1098, (1995)] have observed that the tie-2 promoter, which, in the case of the endogenous gene, is active during vascular proliferation in the embryo or in the adult, behaves differently when it is linked to a heterologous gene; it remains active during the development of the vascularization of the mouse embryo, but is no longer active in the adult, even during angiogenesis.
Using the von Willebrand factor promoter, it was only possible to express the marker gene in some brain endothelial cells; on the other hand, the heterologous gene was expressed in other tissues in which the endogenous gene is not normally expressed [AIRD et al., Proc. Natl. Acad. Sci. USA, 92, p. 4567-4571, (1995)].
Previous research in which the inventors participitated resulted in the isolation of genomic clones containing the gene for VE cadherin and in the determination of the positions of the introns and exons [HUBER et al., Genomics, 32, p. 21-28, (1996)] However, the work did not reveal any sequence controlling the transcription of this gene.